【摘要】 目的 观察牛磺石胆酸(taurocholic acid, TLCA)调节棕榈酸(palmitic acid, PA)诱导的巨噬细胞极化，探讨其对肝细胞脂质代谢的影响。方法 分别采用TLCA、PA处理小鼠单核巨噬细胞(RAW264.7),实验分为对照(CON)组、TLCA组、PA组和PA+TLCA组。24 h后收集各组条件培养基(conditioned medium, CM),分别处理C57 BL/6J小鼠肝脏原代细胞和AML-12肝细胞；使用氨苯蝶啶(triamterene, Tr)抑制巨噬细胞武田G蛋白偶联受体5 (Takeda G-protein receptor 5,TGR5),以证实其在调节巨噬细胞极化以及降低肝细胞脂质沉积中的作用。采用BODIPY荧光染色检测肝细胞内脂质积聚；RT-qPCR检测炎症因子及脂质代谢相关基因mRNA水平；蛋白质免疫印迹法检测巨噬细胞TGR5和肝细胞固醇调节元件结合蛋白-1c(sterol regulatory element-binding proteins, SREBP-1c)表达水平；细胞免疫荧光染色观察细胞内iNOS、CD206蛋白表达。结果 BODIPY荧光染色显示，PA组较CON组肝细胞脂质沉积显著增多，PA+TLCA组脂肪沉积得到缓解(P<0.05);与PA组比较，PA+TLCA组原代肝细胞和AML-12细胞脂质合成基因ACC1、FASN、SREBP-1c的mRNA水平以及SREBP-1c蛋白表达水平显著降低(P<0.05), ACOX1和CPT1A表达水平在各组间无显著变化；RT-qPCR结果显示，PA诱导巨噬细胞向M1型极化，促炎因子(iNOS、IL-6、IL-1β、TNF-α)水平显著升高，而联合TLCA处理后可降低炎症因子水平且增加RAW264.7细胞M2型极化相关基因(Arg1、IL-4、IL-10)mRNA表达水平(P<0.05);与CON组比较，PA组M1型巨噬细胞标志物iNOS显著增加，M2型巨噬细胞标志物CD206明显降低；TLCA可促进TGR5蛋白表达(P=0.071 4),氨苯蝶啶抑制TGR5后，显著抑制TLCA调节巨噬细胞极化和减少肝细胞脂质沉积的作用。结论 TLCA通过巨噬细胞TGR5抑制PA诱导的M1型极化且促进M2型极化，促炎症因子减少，进而降低肝细胞脂质合成，改善肝细胞脂质沉积。更多还原

【Abstract】 Objective To investigate the effect of taurocholic acid(TLCA) regulating palmitic acid(PA)-induced macrophage polarization on hepatocyte lipid metabolism. Methods Mouse mononuclear macrophage(RAW264.7) cells, treated with TLCA and PA, were divided into control(CON), TLCA, PA and PA+TLCA groups. The conditioned medium(CM) was collected for culturing primary hepatocytes from C57 BL/6J and AML-12 liver cells. Triamterene was used to inhibit macrophage Takeda G-protein receptor 5(TGR5), which could investigate the role of TGR5 in regulating macrophage polarization and reducing lipid deposition in hepatocytes. BODIPY fluorescence staining was performed to detect lipid accumulation in hepatocytes; The mRNA levels of genes related to inflammatory factors and lipid metabolism were detected by RT-qPCR; Western blotting was performed to detect the level of protein expression of macrophage TGR5 and hepatic cell SREBP-1c. The expression of iNOS and CD206 were observed by immunofluorescence staining. Results BODIPY fluorescence staining showed that PA group displayed significantly increased lipid accumulation, compared with CON group, while TLCA treatment could reduce the PA-induced lipid accumulation(P<0.05); Compared with the PA group, the PA+TLCA group had decreased mRNA levels of ACC1, FASN and SREBP-1c and protein expression of SREBP-1c(P<0.05). However, there were no significant changes of expression of ACOX1 and CPT1A among these groups mentioned above. The outcome of RT-qPCR showed that PA stimulated RAW264.7 into M1-type polarization and significantly increased the levels of proinflammatory factors(iNOS, IL-6, IL-1β, TNF-α). PA combined with TLCA could reduce the levels of inflammatory factors and increased the mRNA expression levels of M2-type polarization-related genes, Arg1, IL-4 and IL-10(P<0.05). Compared with CON group, the PA group showed increased M1-type marker iNOS and decreased M2-type marker CD206, while TLCA could promote the protein expression of TGR5(P=0.0714). TGR5 inhibited by triamterene could suppress macrophage polarization regulated by TLCA and reduce hepatocyte lipid accumulation. Conclusion TLCA inhibits PA-induced M1-type polarization and promotes M2-type polarization via TGR5, which reduces proinflammatory factors, reduces hepatic lipogenesis and promotes hepatocyte lipid accumulation.更多还原